1. Field of the Invention
The present invention is directed to chromatographic supports for large scale separation and purification of biological macromolecules utilizing various chromatographic separation techniques.
The supports of the instant invention comprise a low molecular weight copolymer covalently bonded to the silica, the low molecular weight copolymer formed from at least one polymerizable compound which has a chemical group capable of direct covalent coupling to said silica particle and one or more polymerizable compounds containing organic functional groups capable of immobilizing a bio-active molecule. In the present invention, the low molecular weight copolymer is first formed and then attached to the silica particle.
2. Brief Description of the Background Art
In chromatographic separation technology, the key factor is the nature of the supporting matrix, which governs capacity, flow speed, and resolution for separation. Resolution of components in a chromatographic column is achieved by partitioning solutes between two physically distinct phases that share a common interfacial boundary, the two physically distinct phases respectively the stationary phase and the mobile phase. During the process of separating the solute mixtures by running the material through the column, the distribution of solute molecules between these two phases is a constant K, usually referred to as the partition coefficient. The partition coefficient for a solute is expressed as K=C.sub.S /C.sub.M, where C.sub.S is the concentration of solute per unit volume of stationary phase and C.sub.M is the concentration of solute per unit volume of mobile phase. Maximum resolution is achieved by choosing the two phases that produce the greatest difference in the partition coefficient of solutes. As is known to the art, the mobile phase can be modified by changing the pH and/or ionic strength of the solvent.
As is also known, the character of the stationary phase plays a substantial role in column performance. In the case of ion exchange, hydrophobic or affinity chromatography, the stationary phase is frequently immobilized on a support matrix. In ion exchange chromatography, the basis for partitioning is ionic association of solutes with anionic or cationic stationary phase bonded to the inert support.
Among the first generation of synthetic ion exchange materials were the ion exchange resins. The fundamental frame work of these ion exchange resins is an elastic three-dimensional hydrocarbon network comprising ionizable groups, either cationic or anionic, chemically bonded to the backbone, a hydrocarbon framework. The network is normally fixed, insoluble in common solvents, and chemically inert. The ionizable functional groups attached to the matrix carry active ions which can react with or can be replaced by ions in the solute phase. Therefore, the ions in the solute phase can be easily interchanged for the ions initially bound to the polymeric resins. Typical examples of commercially available ion exchange resins are the polystyrenes crosslinked with DVB (divinylbenzene), and the methacrylates copolymerized with DVB. In the case of polystyrene, a three-dimensional network is formed first, and the functional groups are then introduced into benzene rings through chloromethylation. Since ion exchange resins are elastic three-dimensional polymers, they have no definite pore size, and therefore only a steadily increasing resistance of the polymer network limits the uptake of ions and molecules of increasing size. The resistance to flow exhibited by these resins is controlled by the degree of crosslinking of the resins. With the low degree of crosslinking, the hydrocarbon network is more easily stretched, the swelling is large, and the resin exchanges small ions rapidly and even permits relatively large ions to undergo reaction. Conversely, as the crosslinking is increased, the hydrocarbon matrix is less resilient, the pores of the resin network are narrowed, the exchange process is slower, and the exchanger increases its tendency to exclude large ions from entering the structure. The ion exchange resins made from polymeric resins have been successfully applied for the removal of both organic and inorganic ions from aqueous media, but they are normally unsuitable for the separation of biopolymers such as proteins. This is due, among other reasons, to the following:
(1) The highly cross-linked structure has rather narrow pores to accommodate the diffusion proteins; the proteins therefore are virtually restricted to the macro surface area of the beads with consequent limitation of solute loadings;
(2) The high charged density close to the proximity of the resin surface is unsuitable, since it causes excessive binding and distortion of protein structure;
(3) The hydrocarbon matrix is usually hydrophobic and is potentially damaging to the subtle three-dimensional structure of biopolymers, often causing denaturation of proteins.
The above limitations fostered the next generation of chromatographic materials useful for separation of proteins and other labile biological substances. This next generation of chromatographic materials was based on cellulose ion exchangers. The cellulose ion exchange materials have been made by attaching substituent groups with either basic or acidic properties to the cellulose molecule by esterification, etherification, or oxidation reactions. Examples of cationic exchange celluloses are carboxymethylated cellulose (CM), succinic half esters of cellulose, sulfoethylated cellulose, and phosphorylated cellulose. Examples of anionic exchange celluloses are diethylaminoethyl cellulose (DEAE), and triethylaminoethyl cellulose (TEAE). However, the ion exchange materials based on cellulose as the principle backbone or anchoring polymer have not enjoyed complete success due primarily to an inherent property of cellulose, its affinity for water. Thus, prior art ion exchange materials based on cellulose, while typically having high exchange capacity, are difficult to use as a consequence of their tendency to swell, gelatinize, or disperse on contact with an aqueous solution. And while these cellulosic ion exchangers are advantageous in that they lack nonspecific adsorption and have practicable pore structure, an ideal ion exchange material should only minimally interact with the solvent system which carries the ion in solution through its pores. This at most minimum interaction with the solvent system is essential to obtain a rapid, free-flowing ion exchange system.
Perhaps the most popular inner support used in the laboratory for chromatographic separation of proteins, nucleic acids, lipoproteins, peptides, and vitamins are the polysaccharides such as Sephadex.RTM. and Sepharose.RTM.. The success of polysaccharides as chromatographic support is primarily based upon the ability of saccharides to imbibe large quantities of water and swell into a hydrophilic matrix, the chemical stability of the hydrophilic matrix, the relative ease with which these polysaccharides can be derivatized with functional groups, and the ability of saccharides to stabilize the sensitive biological molecules.
However, these polysaccharide matrices lack mechanical strength and are extremely sensitive to change in pH, ionic strength, and pressure. These limitations become especially acute for large scale operations where it is desirable to use forced flow and drastic change of pH and ionic strength in the process of separation. Sephadex.RTM. exhibits very low non-specific adsorption, thereby making it ideal as a matrix for biological separations. However, the porosity of ion exchange gels is critically dependent on its swelling properties, which in turn are affected by the environmental ionic strength, pH, and the nature of the counter-ions.
Swelling of gels in buffers is caused primarily by the tendency of the functional groups to become hydrated. The amount of swelling is directly proportional to the number of hydrophilic functional groups attached to the gel matrix, and is inversely proportional to the degree of crosslinking present in the gel. This characteristic swelling is a reversible process, and at equilibrium there is a balance between two forces: the tendency of the gel to undergo further hydration, and hence to increase the osmotic pressure within the gel beads, and, in opposition, the elastic forces of the gel matrix. Since the pH, the electrolyte concentration, and the nature of counter-ion can all affect the hydration, leading to a different degree of gel swelling, the pore size in the gels is not well defined but is rather dependent upon environmental conditions. Gels without crosslinking provide large pores and high capacity due to maximum swelling. However, the same non-crosslinked gels lack structural integrity and are easily crushed with a minimum amount of pressure. Removal of the solvent from the gels often results in the collapse of the matrix. In contrast, highly crosslinked gels have sufficient mechanical strength, but lose capacity and pore size due to restriction in swelling.
Also known to the prior art are ion exchange gels made from synthetic polymers. These include crosslinked polyacrylamide (Bio-Gel P.RTM.), microreticular forms of polystyrene (Styragel.RTM.), polyvinyl acetate (Merck-o-Gel OR.RTM.), crosslinked poly (2-hydroxy-ethylmethacrylate) (Spheron.RTM.), and polyacryloylmorpholine (Enzacryl.RTM.). With each of these, it is possible to obtain either dimensional stability without high capacity, or high capacity without dimensional stability. It is, however, not possible to obtain both high capacity and dimensional stability at the same time.
This failure of a single component to demonstrate both high capacity and dimensional stability has led to yet another generation of ion exchange materials comprising composite structures, hybrid gels. Hybrid gels are made by combining a semi-rigid component, for the purpose of conferring mechanical stability, with a second component, a softer network, which is responsible for carrying functional groups. Agarose gel, which would otherwise be very soft and compressible, can be made stronger by hybridizing with crosslinked polyacrylamide. The crosslinked polyacrylamide component is mechanically stronger than the agarose, improves the gel flow properties, and reduces the gel swelling, but it sacrifices molecular fractionation range. Other hybrid gel examples include polyacryloylmorpholine and agarose, as well as composite polystyrene gels, large pore polystyrenes as a frame work, filled with a second type of lightly crosslinked polymer.
Yet another composite gel structure is achieved by combining inorganic materials coated with organic materials, typically the type known as Spherosil.RTM.. Porous silica beads are impregnated with DEAE-dextran so that the product will have the mechanical properties of silica, with the ion exchange properties of DEAE-dextrans. These composites, however, have severe channeling defects arising as a result of particle packing, and they have capacity limitations on the coated surfaces.
Totally rigid inorganic supports such as porous silica or porous glass which are susceptible to degradation have also been used to provide high porosity, and high flow rate systems. However, non-specific adsorption of proteins due to the silanol groups on the silica surface is a problem. Since the hydrolysis of silica is directly related to the pH conditions, the non-specific adsorption by silica is minimal at neutral pH, but increases as the pH changes to the acidic or alkaline ranges. Prior art methods, to avoid the non-specific adsorption of silica as a result of pH changes, have involved coatings of hydrophilic organic polymers. However, these prior art coating methods have relied heavily on silane coupling agents. Structurally, the silane coupling agents have the general formula X.sub.3 --Si--RY, where Y is the organic functional group and X.sub.3 are hydrolyzable groups. The organic functional groups (Y) are chosen for reactivity or compatibility with the bio-macromolecules, while the hydrolyzable groups are intermediates in formation of silane groups for binding to mineral inorganic surfaces. Typical silane monomers which are specifically useful for chromatography have the formula (CH.sub.3 O).sub.3 SiCH.sub.2 R in which R could be amino, DEAE, or glycido (epoxy).
U.S. Pat. No. 3,983,299 to Regnier, and U.S. Pat. No. 4,029,583 to Chang et al., both assigned to Purdue Research Foundation, disclose the use of glycidoxypropyltrimethoxysilane attached on a silica support. Basically, the trimethoxy groups react with the available hydroxy groups on the silica particles leaving the oxirane ring available for later opening and attachment of the nucleophillic stationary phase. However, this method has proven to have little commercial value as the capacity of coating adhesion has been found to be rather poor.
Boardman, N. K., J. Chromatog. 2, pages 388 and 389 (1959) discloses formation of a thin layer of resin in the cavities of an inner porous support by precipitation copolymerization of suitable monomers in methanol solution in the presence of celite. A weak cationic exchanger which had a capacity of 0.69 meq/g was made from methacrylic acid and divinyl benzene. For preparation of a strong cationic exchanger, styrene and 5% divinyl benzene were copolymerized to a silanol-treated celite, the product subsequently sulfonated. The capacity of this resin was 0.28 meq/g. Since the resin layer is only deposited or coated on the silica surface, the bonding between the polymer and the silica is quite weak and the capacity quite low. U.S. Pat. No. 3,577,266 to J. J. Kirkland discloses a pellicular silica support wherein a porous silica is impregnated with a suitable monomer mixture containing an initiator, a crosslinked polymer being formed in the cavities of the silica gel in situ. Either an ionogenic monomer is used or the ionic groups are introduced into the polymer by subsequent chemical reaction.
Kalal et al., U.S. Pat. No. 4,332,694 discloses three-dimensional carriers of inorganic porous material (silica) in combination with a reactive polymer containing epoxy groups, a part of the epoxy groups reacting with SiOH groups on the surface of the inorganic porous material, a part of the epoxy groups available for oxidation, isomerization, or hydrolysis to effect substitution of functional groups. Kalal et al. does not distinguish between in situ polymerization and treatment of the inorganic substrate with a low molecular weight polymer containing the epoxy groups. Further, the reference does not recite modifiers which are copolymers of a reactive monomer capable of covalently bonding to the substrate and a different linking monomer capable of immobilizing biological materials.
A carrier for bioactive material is also disclosed in Nakashima et al., U.S. Pat. No. 4,352,884. The Nakashima carrier comprises a substrate coated with a copolymer. The substrate may be one of various materials, including inorganic materials such as silica. The copolymer is made of a hydrophilic acrylate or methacrylate monomer which is a hydroxy or alkoxy acyl acrylate or methacrylate, and a copolymerizable carboxylic acid or amine. The base material or substrate is coated with the copolymer with conventional coating or deposition procedures, such as spraying, dipping, phase separation or the like. The copolymer may also contain small amounts of a crosslinking agent such as glycidyl acrylate or methacrylate. The crosslinking agent allows for crosslinking treatment after the coating process and provides for the prevention of elution (presumably of the bioactive materials) from the coating layer. The amounts of crosslinking agent are quite small, they range between 0.5 and 1% by weight of the total copolymer weight. Such amounts of crosslinking agent are insufficient to cause substantial covalent bonding or grafting of the copolymer onto the underlying substrate. The copolymer in Nakashima is thus essentially only physically coating the underlying substrate. Physical coating, however, is accompanied by a series of problems. The carrier would not be expected to have an even distribution of the copolymer which shows a multi-layered structure, and may have a possible uneven distribution of functional groups.
Another reference of interest is Kraemer, U.S. Pat. No. 4,070,348, which shows copolymers of glycidyl- and amino-containing acrylates, useful as carriers for biologically active substances such as polysaccharides, enzymes, peptides, hormones, etc. The structure of the final product, in Kraemer is that of an acrylic copolymer chain covalently modified at a multiplicity of sites thereon with substances such as enzymes, proteins, and the like. In contrast thereto, in the instant invention the silica particles are coated with a plurality of acrylic copolymer chains, each chain covalently attached on the silica particle.
Good, U.S. Pat. No. 3,808,125, provides a chromatographic material which may be a packing material such as silica, the packing material having a stable uniform film of a polymeric stationary phase. The stationary phase is chemically bonded to the material, either directly or through a chemical coupling or bonding agent which has a reactive group capable of reacting with the reactive site provided by the silica, and also a reactive site capable of reacting with the polymeric agent. Typical polymeric materials described by Good are those materials derived from compounds having the formula CH.sub.2 .dbd.C(R).sub.2 wherein R can be --COOR.sup.2, wherein R.sup.2 may be hydrogen. Thus the polymeric film covalently coupled to the silica of Good is a polyacrylic or a substituted polyacrylic acid or other unsaturated acid. The coupling agents to be used to combine the polyacrylate to the silica must be capable of forming an integral physical or chemical bond to the polymeric film (see col. 8, lines 45-48) and are generally described as being unsaturated silanes (see col. 8, lines 70ff). Thus silica is chemically bonded to a "copolymer" made from the coupling agent polymerized onto a homo- or copolymer which may be polyacrylate or polymethacrylate. These copolymers do not carry ionic exchange groups or anchoring groups for affinity chromatography as described in the instant invention.
Kirkland et al., U.S. Pat. No. 3,795,313, at example 3, describes a siliceous material coated with a methacryloxy silane. The compound is not further reacted in any fashion in this patent and no suggestion is made of preparing polymers or copolymers bonded thereto.
Fuller, U.S. Pat. No. 3,878,092 describes silica coated with a polymer containing "at least one divinyl aromatic hydrocarbon or at least one monovinyl aromatic hydrocarbon"-derived polymer.
Thus, in spite of the vast amount of research endeavor in the area of supports for carriers for immobilization of physiologically active materials, a need has continued to exist for chromatographic supports for large scale separation and purification of biological macromolecules which have sufficient mechanical stability to withstand high flow velocity and pressure, demonstrate highly specific adsorption at various pH conditions, provide sufficient porosity to allow fast penetration of and partitioning of biological molecules through the supports, demonstrate sufficient hydrolytic stability of the bonded stationary phase and adequate hydrophilicity to permit extended operation in aqueous systems, are inexpensive to produce (the use of silane coupling agents is extremely expensive), and have a high capacity as the result of the introduction of substantial numbers of functional groups on the silica surface, thereby leading to higher column capacity.